Amperometric detection in TMB/HRP-based assays (original) (raw)

Abstract

3,3′,5,5′-Tetramethylbenzidine (TMB) is the most commonly used chromogen for horseradish peroxidase (HRP) and so its performance as an electrochemical substrate was evaluated. Measurements of HRP activity in solution were carried out by using an amperometric detector coupled to a flow injection analysis (FIA) system. The enzymatic product was easily detected at a potential of +0.1 V (vs. Ag-pseudoreference electrode) at a bare screen-printed electrode placed in a homemade electrochemical flow cell. A high flow rate (4.3 mL min−1) of 0.5 M H2SO4 was used to obtain repeatable signals and a short analysis time. The detection limit achieved after 15 min of incubation was 2×10−14 M of HRP. The applicability of the amperometric detector to ELISAs was demonstrated by using a commercially available kit for the quantification of interleukin-6 (IL-6) without modifying the kit manufacturer’s protocol or the reagents for this test.

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Acknowledgements

This work has been supported by the Project BIO2003-06008-C03-01.

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Authors and Affiliations

  1. Departamento de Química Física y Analítica, Universidad de Oviedo, 33006, Oviedo, Asturias, Spain
    Pablo Fanjul-Bolado, María Begoña González-García & Agustín Costa-García

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  1. Pablo Fanjul-Bolado
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  2. María Begoña González-García
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  3. Agustín Costa-García
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Correspondence toAgustín Costa-García.

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Fanjul-Bolado, P., González-García, M.B. & Costa-García, A. Amperometric detection in TMB/HRP-based assays.Anal Bioanal Chem 382, 297–302 (2005). https://doi.org/10.1007/s00216-005-3084-9

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